Method of removing hepatitis-associated antigen from a protein fraction using polyethylene glycol

ABSTRACT

A METHOD OF REMOVING HEPATITIS-ASSOCIATED ANTIGEN FROM A PROTEIN FRACTION BY REGULATING PROTEIN CONCENTRATION, IONIC STRENGTH OF THE SOLUTION, AND PH, AND FRACTIONATING THE MIXTURE BY THE USE OF POLYETHYLENE GLYCOL IN A PARTICULAR CONCENTRATION RANGE IS DISCLOSED. THE PH OF THE SOLUTION SHOULD BE REMOVED FROM THE ISOELECTRIC POINT OF THE MATERIAL BEING PRECIPITATED.

United States Patent 3,790,552 METHOD OF REMOVlNG HEPATITIS-ASSOCI- ATEDANTIGEN FROM A PROTEIN FRACTION USING POLYETHYLENE GLYCOL Alan J.Johnson, New York, and Jack Newman, Pelham, N .Y., assiguors to theUnited States of America as represented by the Secretary, Department ofHealth, Education, and Welfare No Drawing. Filed Mar. 16, 1972, Ser. No.235,211 Int. Cl. A23j 1/06; C07g 7/00 US. Cl. 260-112 B 28 ClaimsABSTRACT OF THE DISCLOSURE A method of removing hepatitis-associatedantigen from a protein fraction by regulating protein concentration,ionic strength of the solution, and pH, and fractionating the mixture bythe use of polyethylene glycol in a particular concentration range isdisclosed. The pH of the solution should be removed from the isoelectricpoint of the material being precipitated.

BACKGROUND OF THE INVENTION This invention relates to the separation ofblood proteins, and more particularly this invention relates to theremoval of hepatitis-associated antigen from other protein fractions.

Hepatitis is a relatively common disease, but the disease is sometimesdiflicult to diagnose and there is, as yet, no specific treatment. Eventhough as many as five percent of the reported cases become chronicallyill and another two percent become cirrhotic, it has been estimated thatthree out of every thousand persons who become infected with the serumhepatitis virus do not become ill, but nevertheless carry the disease.For this reason hepatitis poses a serious problem in detecting blooddonors who may transmit the disease.

The known methods for detecting serum hepatitis rely on detection ofvirus products in the body fluids but are time-consuming and burdensomeas well as being rela tively insensitive. In fact, it has been estimatedthat most of the prior art tests can detect only twenty to thirtypercent of the carriers of the disease. Moreover, prior tests have notbeen sensitive enough to detect antibodies that develop after a singleexposure to hepatitis virus or primary hepatitis infection.

A specific antigen, popularly known as Australia antigen has been foundin the serum of many patients with serum hepatitis. The exact pathogenicrole of the Australia antigen is a current problem of great academicinterest as well as urgency in view of the importance of recognizing thesource and/or cause of hepatitis. The Australia antigen is also known ashepatitis-associated antigen (HAA), which terminology will be usedherein. Most of the common methods used for the detection of hepatitisrely on the presence of the HAA, or the antibody thereto, for theirspecificity.

At present, the neutralizing effect of immune serum, that is, the serumof an individual who has recovered from hepatitis and containsantibodies specific against the disease, is used to diagnose hepatitisby various tests. In these tests HAA antibody is added to the same,forming a precipitate or complex with the antigen present in the sample.

Existing techniques for detecting and diagnosing hepatitis include:irnmunodiffusion, complement fixation, electrophoretic modifications ofprecipitin techniques, radioimmunoassays, and hemagglutinationprocedures. Diffusion methods are the simplest but the least sensitiveand slowest. The electrophoretic modifications of precipitin techniquesare rapid, as rapid as complement fixation, but

3,790,552 Patented Feb. 5, 1974 not quite as sensitive. Of the availabletechniques, complement fixation and hemagglutination procedures appearto be the most rapid and are also moderately sensitive. The complementfixation technique involves adding a biologic material such as blood orplasma to the appropriate antibody for HAA. The reaction mixture isincubated with a predetermined amount of complement to fix the latter.The amount of HAA or antibody in the body fluid can be determinedthrough titrating the amount of remaining non-fixed complement byincubation with a standard cell solution. The complement fixationtechnique is only moderately sensitive for detecting antigen andantibody, gives anti-complementary reactions with plasma fractions, andis not routinely available in many hospitals and blood banks. Thevarious radioimmunoassays used are the most sensitive tests available atpresent but usually require at least one day to run, and thereproducibility is only fair. The hemagglutination test withantibody-coated red cells appears to combine both sensitivity andrapidity.

Epidemiological surveys following transfusion therapy reveal a very highincidence of hepatitis, in the order of 0.l2l2 percent with pooledplasma or whole blood. The incidence of hepatitis with fibrinogentransfusion has been variously estimated at nine percent to more thanthirtysix percent, whereas one investigator believed the incidence wasthirty-five times as high as with transfusion of blood or plasmafractions. The fibrinogen is considered to be contaminated because it isisolated from very large plasma pools (5000 or more units, some of whichcontain HAA) but probably contains only about one percent of thehepatitis-associated antigen present in the original plasma. Thetendency to transmit hepatitis has severely limited the usefulness offibrinogen or fibrinogen-containing fractions, e.g., when Fraction 1-0containing sixty to eighty percent fibrinogen was used as a source ofAHF in this country, it caused hepatitis in severty-five percent ofhemophiliacs not previously given massive transfusions of fresh-frozenplasma and in fifty percent of those who were previously transfused. Itis estimated that more than ten percent of patients who develophepatitis die from its effects.

Prior screening of all blood donors by present assays will probablyreduce the incidence by only twenty to thirty percent since theavailable assays for the antigen associated with serum hepatitis (HAA)are relatively insensitive and plasma with barely detectable HAA contentby in vitro assay usually produces overt disease. Furthermore, injectionof a 1,000,000 dilution of such plasma may cause demonstrable viremiawithout icterus. Since professional blood donors, who have a ten timeshigher incidence of hepatitis than volunteer blood donors, furnishalmost half the countrys blood supply, it would be very difiicult toexclude them as donors and indeed would be unnecessary if the HAA couldbe removed during blood collection. Patients receiving transfusionscould be given ordinary gamma globulin but it has proved to be helpfulonly against infections hepatitis; or they could be given high-titerimmune gamma globulin from recovered hepatitis patients, but the supplyis exceedingly limited and its usefulness in preventing clinical diseasein patients infused with large amounts of HAA contaminated plasmafractions has not yet been proved conclusively.

The HAA or serum hepatitis (SH) antigen probably participates in thepathogenesis of serum hepatitis, and is viewed primarily as a marker inplasma for the disease. The HAA particles found in infected blood arepredominantly of two morphologic types: those about 22 my. in diameterwhich seem to represent incomplete virions (empty capsids), andparticles about 42 me in diameter which may represent the entire virus.

For many years the risk of hepatitis has prevented the widespread use offractions such as fibrinogen and slowed the commercial production of aconcentrate of Factors II, VII, IX and X as well as Factor VIII(antihemophilic factor-AI-I F), from large pools of human blood. Becausethe Cohn procedure has been widely used for many years and little or noserum hepatitis develops after treatment with Cohn Fraction II andalbumin, the method has been considered more or less sacred ordefinitive."

These considerations have mitigated against the commercial use of newfractionation methods such as the zinc method XIII and DEAE adsorptionin the purification of gamma globulin, and polyethylene glycol (PEG) inthe purification of serum albumin although these methods wouldundoubtedly result in products of higher purity and yield, loweraggregation potential and greater stability. Even new anticoagulantssuch as citrate phosphatedextro'se (CPD) cannot be used without priorextensive clinical trials of each plasma fraction derived from bloodcollected with CPD and fractionated by the Cohn proce dure, to ascertainits potential for transmitting hepatitis a long, tedious, expensive andtime-consuming process. Thus, in addition to demonstrating that newfractionation techniques or use of CPD blood result in plasma fractionsof higher purity and yield than the Cohn fractions, it is necessary toshow that they are no more likely to cause hepatitis.

Some therapeutically valuable plasma fractions are often contaminatedwith HAA. In the hands of most investigators, commercially preparedconcentrates of Fractors II, VII, IX and X have been associated with ahigh incidence of hepatitis, at least seventeen percent, while one studyreports forty to seventy percent. This material is therefore reservedprimarily for the treatment of a small group of bleeding patients withFactor IX deficiency and a history of multiple transfusions. Many otherbleeding patients defincient in one or more of the four factors but withno history of transfusions would probably benefit greatly from therapywith plasma, plasma fractions, and whole blood free of HAA. This groupincludes patients with hepatitis, cirrhosis, liver poisoning or otherparenchyrnal liver damage, or with dicumarol overdosage or biliaryobstruction.

Possible HAA contamination of AHF concentrates and fibrinogen also seemsto be a pressing problem. Recent emphasis on HAA by the Division ofBiologics Standards (DBS) has pointed up the need for screening donorsbefore the plasma is fractionated as well as three months after bloodcollection to make sure that they are still free of the antigen. It isvery difficult, however, to predict whether donors will developHAA-positive blood six weeks to three months after giving blood.According to recent estimates, a fairly high percentage do so. It isalso diflicult to rule out circulating antigen-antibody complexes thatgive negative results with present HAA assays and may subsequently proveto be infective.

The high morbidity and mortality attending post-transfusion hepatitiscan probably be reduced to some extent by modifying present plasmafractionation methods. About 50,000 units of unselected plasma werefractionated from 1966 to 1969 to produce American National Red Cross(ANRC) intermediate-purity AHF which was used with other materials totreat 153 episodes in 96 patients with hemophilia .A or von Willebrandsdisease. Although some of them received very large amounts, only twodeveloped hepatitis and they had also received large amounts of otherFactor VIII concentrates and plasma. Similarly, about 50,000 units ofunselected plasma were processed for the ANRC high-purity AHF which wasused with other Factor VIII preparations to treat 92 episodes in 57patients. One of these developed hepatitis and he, too, had receivedlarge amounts of other AHF-containing materials. Since other patientsgiven intermediateor high-purity AHF from the same lots did not developthe disease, the level of any contaminating HAA present in thepreparations seems to have been very low indeed.

In order to prevent transmission of hepatitis by blood donors, mosttransfusion centers are setting up one of the new assay methods fordetecting HAA in the donors blood. If it is found, the HAA-positiveplasma is usually sterilized and discarded. As mentioned above, thesemethods will detect the HAA in only 20 to 30 percent of the contaminatedplasma units.

Polyethylene glycol in the fractionation and concentration of proteinsand viruses: Albertson used polythylene glycol (PEG) to fractionate andconcentrate cells, viruses, microsomes, proteins, nucleic acids, andantigen-antibody complexes in two-phase systems with dextrans orammonium sulfate and water. This synthetic, non-reactive polymer hasalso been used as a vehicle for intramuscular and intravenousadministration of various hormones. Polson et a1. fractionated albumin,gamma globulin and fibrinogen with PEG of 6000 molecular weight, and aprocedure for the preparation of high-purity AHF with PEG of 4000 and6000 M.W. is described and claimed by Johnson et al. in co-pending US.patent application Ser. No. 663,507, filed Aug. 28, 1967, now 'U.S. Pat.No. 3,652,530. In general, PEGs mode of action is believed to be basedon removal of water from the hydrophilic region of proteins; as aresult, their hydrophobic bonds presumably interact causing aggregationand a decrease in solubility.

Extensive acute and chronic toxicity studies at the Mel- Ion Institutefrom 1947 to 1970, principally with PEG- 4000, further attested thenontoxic nature of the polymer. In animals, intravenous doses as high as16 gm./kg. were given to four species without evidence of toxicity. In52 hemophiliacs and Von Willebrand patients, high-puriyt AHFprecipitated with PEG was administered during 92 treatment episodes withno toxic effects. PEG is rapidly excreted by the kidneys, with aglomerular clearance rate virtually the same as that of inulin andcreatinine.

PEG has also been used by virologists to concentrate and purify a numberof plant viruses, bacteriophages, and some animal organisms:

(a) Hebert precipitated partially purified soil-borne wheat mosaic virus(WMV) from a growth medium with three percent PEG-6000 andcentrifugation for ten minutes at 10,000 r.p.m. In other studies, heprecipitated rodshaped WMV particles in 0.1 M phosphate buffer at pH 7.5with two percent PEG. Tobacco mosaic virus (TMV) was also precipitatedfrom extracted, infected tobacco leaves by four percent PEG in 0.1 MNaCl and by two percent PEG in 0.3 M NaCl. In contrast, eight percentPEG was required to precipitate two plant viruses with sphericalparticles: tobacco ringspot virus (precipitated by treating the juicewith PEG in 0.3 M NaCl) and the bean pod mottled virus (precipitated in0.2 M NaCl).

(b) When purifying African horse-sickness virus for electronmicroscopy,Polson and Becks precipitated the virus from emulsified mouse braintissue using three to four percent PEG-6000 and 0.066 M phosphate bufierat pH 7.0.

(c) Leberman described precipitation of four highly purified virusesfrom solutions: turnip crinkle virus (TCV), turnip yellow mosaic virus(TYMV), a nitrous acid mutant of tobacco mosaic virus (TMV), andbacteriophage T-4 with PEG-6000 at varying pH and NaCl concentrations.Similar studies were later carried out with only partially purifiedvirus solutions. Precipitation with PEG required a higher N aClconcentration as the pH was raised.

(d) In earlier studies, viruses with a nucleic acid core and a proteincoat were precipitated with PEG. However, McSharry and Benzinger appliedthe method to a virus with a lipiprotein coat: vesicular stomatitisvirus. The

virus was effectively precipitated with a six to eight percentconcentration of PEG-6000 and 0.5 M NaCl; these researchers concludedthat PEG precipitation concentrates and purifies representatives of allmajor virus classes except the pox-virus and herpesvirus groups (not yettested) without loss of infectivity.

(e) In experiments by Yamomoto et al., seven bacteriophages were readilyconcentrated from crude lysates of infected bacteria by two to tenpercent PEG-6000 and 0.5 M NaCl. The efiicacy of the method wasrelatively unaffected by changes in pH ionic strength. They also showedthat the asymmetric particles of TMV and bacteriophage fd wereespecially susceptible to PEG and could easily be purified from the moresymmetric phage particles at low PEG concentrations. In addition, theyfound that a relatively constant percentage of phage was precipitated bya fixed concentration of PEG over a phage concentration range of nearlyand concluded that the exact mechanism of precipitation is unknown but aphase partition rather than a normal precipitation reaction seems to beinvolved.

(f) Juckes precipitated proteins with PEG-6000 in an effort to determinethe mechanism (5) involved. He varied the pH, ionic strength, proteinconcentration, and temperature, and concluded that each variable had aneifect on precipitability of the proteins, studied: carboxyhemoglobin,ovalbumin and bovine serum albumin, as well as bromegrass mosaic virus.He concluded that precipitability was related primarily to the molecularweight of the protein and the pH, as measured by difierences in theStokes radius of the proteins, but could not relate the other effects tochanges in the Stokes radius. These principles did not obtain whenviruses and other proteins in low concentration and at low ionicstrength were precipitated.

(g) Pert reported that antibody-antigen reactions can be enhanced withprotein-precipitating agents such as PEG. In one phase of theseexperiments, he added eight percent PEG to eliminate fibrinogen, alphamacroglobulin, cryoglobulins and some other large-molecular-weightproteins, then used twelve percent PEG to precipitate and concentratemost of the HAA together with many other moderate-sized proteins such asgamma globulin, alpha 1 and alpha 2 globulins, beta globulins, and theirassociated lipiproteins.

SUMMARY OF THE INVENTION Since the heptatitis-associated antigen (HAA)has been associated with, and may be involved in the pathogenesis of,serum heptatitis, absence of the antigen has been interpreted asindicating relative freedom from the infectious agent causing serumhepatitis. The primary object, therefore, is to provide a method toeliminate the antigen from ful plasma fractions-albumin, gammaglobulins, coagulation Factors II, V, VII, IX, X, XI, XII and XHI,fibrinoful plasma fractions-albumin, gamma globulins, coagulationFactors II, V, VII, IX, X, XI, XII and X11, fibrinogen, antihemophilicfactor (Factor VIII), plasminogen, ceruloplasmin, transferrin,thyroxin-biding protein, antithrombin HI, cal antitrypsin, a2macroglybulin. Ci inactivator, inter-u trypsin inhibitor, as well assewage-by fractionation with polyethylene glycol (PEG).

It is another primary object of the prsent invention to provide a methodfor removing the contaminating HAA from plasma fractions that have beenpartially purified.

It is still another object of the present invention to provide asuperior fractionation method using an agent which does not denature orcombine with plasma proteins, but selectively concentrates the HAA fromalbumin, gamma globulin, and fibrinogen, as well as concentrates of AHFand Factors II, VII, DC, and X. Variations of this method are used toremove the antigen from albumin, gamma globulin, fibrinogen, aconcentrate of Factors H, VH, and IX, and a concentrate of Factors II,VH, IX and Xall prepared for therapeutic purposes.

In accordance with the foregoing objects, a method is provided whichcomprises the essential steps of: (1) maintaining the solubility of theHAA-containing protein fraction at a pH away from its isoelectric point,(2) adding polyethylene glycol with a molecular weight ranging fromabout 200 to 6000 to a concentration of from about 12 to about 30percent to thereby precipitate the HAA, and (3) separating the HAA fromthe protein.

The major factors that are varied to remove the HAA from the variousfractions are the final PEG concentration of the mixture, the pH, theionic strength, and the protein concentration. The PEG concentration isvaried from about 12-30 gm./ ml. of solution, depending on the molecularweight of the polymer. The pH is adjusted so that it is removed as faras possible from the isoelectric point of the fraction remaining insolution, but without denaturing the protein and still within theprecipitability range for the HAA. The pH is generally separated by 1.0or 2.0 pH units from the isoelectric point, which is well known for mostof the protein fractions and in any event can be easily determined byone of ordinary skill in the art, using known methods. As examples, theisoelectric point of fibrinogen is about 5.5, of gamma globulin from 6.5to 7.5, nominally 7.2, of albumin about 4.9, of Factor 1X about 4.3, andFactor 11 about 4.7.

The solutions are made with any suitable buffer or the like, providedthat the small amounts which may be carried along in the fractionationprocedure are physiologically tolerable 0n I.V. or LP. injection.Typical suitable materials for these solutions are a glycine-citratebuifer, a phosphate buifer, a saline solution, a tris-citrate bufferwith or without other additives such as urea, alone or in variouscombinations. The ionic strength may vary from 0.20 to 0.001.

After the HAA is precipitated, it is removed from the supernatant by anysuitable procedure such as centrifugation or filtration or both. Thefilter should have pores of less than 0.6 mg, preferably between about0.45 and 0.2 mil. Suitable filters are commercially supplied byMillipore or Cox.

Temperature is not a factor in the method of the present invention,although for practical purposes it may be conveniently practiced at roomtemperature or from about 15 to 25 C. At lower temperatures, lowerconcentrations of PEG are used.

While the method of the present invention is preferably practiced byselectively precipitating the HAA to re move it from a solution of thesame with another protein fraction, it should be understood that all theproteins could be precipitated by PEG and the fraction to be separatedfrom the HAA then solubilized with a suitable buffer at a proper pH.

The foregoing objects of the present invention and other objects will inpart be obvious and in part be pointed out as the description of theinvention proceeds.

DESCRIPTION OF THE PREFERRED EMBODIMENT Concentration of HAA fromfractions for assay One milliliter of serum or partially purified HAA(isolated and washed by centrifugation) with an HAA concentration barelydetectable by counter-immunoelectrophoretic assay, was diluted up to10,000 times with fibrinogen, albumin, or gamma globulin, prepared byCohn methods 6 and 9, or purified concentrates of Factors 11, VII, IXand X or Factor VIII. The added HAA was precipitated from the plasmafraction by PEG, reconstituted to its original volume in buffer andassayed (Table 1). Since only a minute amount of precipitate wasobtained, it was collected by batch centrifugation at moderatespeedabout 10,000 relative centrifugal force TABLE 1 RECOVERY OFUNDETECTABLE AMOUNTS OF HAA FROM ALBUMIN BY PRECIPITATION ANDCONCENTRATION WITH POLYETHYLENE GLY- COL (PEG) PEG fraction: Percent HAArecovered Supernatan (containing albumin) Precipitate 100 *One ml. orpartially purified HAA, positive on counterimmuno-electrophoresis at adilution of 1 35, proved negatwo by HAA assay when diluted with 2000 ml,of five percent human serum albumin.

Removal of HAA from fractions by PEG precipitation In other experiments,HAA-rich serum or a serum fraction (complement-fixation titer over 1000)or partially purified HAA (isolated and washed by ultra-centrifugation)was mixed with a partially purified concentrate of Factors II, VII, IXand X prepared by DEAE adsorption of plasma and subsequent elution, orwith AHF prepared by cryoethanol precipitation, or with fibrinogen,albumin or gamma globulin prepared by Cohn methods 6 and 9. The addedHAA was precipitated from the plasma fraction by PEG, and the desiredfraction itself was then precipitated and concentrated by PEG. The yieldof the plasma fraction was 90-100 percent for all except fibrinogen andAHF (Tables 2-4).

TABLE IL-REMOVAL OF HAA FROM A MIXTURE OF THE ANTIGEN AND A CONCENTRATEOF COAGULATION FACTORS II, VII. IX AND X BY POLYETHYLENE GLYCOL (PEG)PRECIPITATION TABLE III-REMOVAL OF HAA FROM A MIXTURE OF THE ANTIGEN ANDHUMAN ALBUMIN BY POLYETHYLENE GLYCOL (PEG) PRECIPITATION HAA titer Totalprotein (countenmmnnoelectro- Fractionation step Mg. Percent phoresis)tate=final product) 2, 900 93 b Neg. 2d PEG supernatant (discard) 130 4b Neg.

e Complement fixation titer-1/410. b Neg. undilute.

1 RCF: e2 2 X gravity wherein rzradius (in feet) of the rotor andn=revolutions per second=r.p.m. 60.

TABLE IV.REMOVAL OF HAA FROM A MIXTURE OF THE ANTIGEN AND HUMAN GAMMAGLOBULIN BY POLY EIHYLENE GLYOOL (PEG) PRECIPITATION HAA titer Totalprotein (counterimmunoelectro- Fractionation step Mg. Percent phoresis)Gammaglobulin plus HAA (starting material 546 1 1/24 1st PEG precipitate(discard) 11.1 2 1/20 2d PEG precipitate (solubilized pr cipitate =fina1product) 507 93 b Neg. 2 PEG supernatant (discard) 26 5. 1 b Neg.

! Complement fixation titer-U614. b Undilute.

EXAMPLE 1 Concentration of HAA from a plasma fraction containingprothrornbin complex (Factors II, VII, IX and X) and preparation of thefraction free of hepatitis-associated antigen (HAA) for clinical use Thestarting material commonly used for this procedure may be partiallypurified prothrombin complex obtained by DEAE-Sephadex or DEAE columnchromatography or from calcium phosphate adsorption and elution methods.The ionic strength for an Optimal yield of these fractions ranges from0.15 to 0.001, and the protein concentration ranges from 3 to 27 mg./ml.at about neutral pH, with water and a suitable buffer, e.g., sodiumcitratesodium chloride. The temperature of this solution is maintainedat 15-25 C. Polyethylene glycol (PEG), molecular weight 200-6000, isadded to a final concentration of 12-30 grams per 100 ml. The exactpercentage is increased for PEG of low molecular weight and decreasedfor the high molecular weight material. After sufi'icient mixing todissolve the PEG and precipitate the HAA, usually 30 minutes, thesolution is centrifuged at room temperature for at least 10 minutes at arelative centrifugal force (RCF) of approximately 10,000 and thesupernatant in separated cleanly from the precipitate by decantation orvacuum aspiration and reserved.

The HAA precipitate is reconstituted with three volumes of buffer orsaline solution, and the walls of the centrifuge bottle are washed,bringing the total volume to about 10 percent of the starting material,or to a volume which readily permits transfer of the reconstitutedprecipitate from the large centrifuge tube to a small one. The HAA isthen reprecipitated from the buifer or saline solution with 12-30percent PEG 200-6000 and centrifuged. Since the precipitate contains theHAA, special care must be taken in this separation and in disposition ofthe precipitate after it has been reconstituted in 0.2-1.0 ml. of wateror saline solution and assay. This optional procedure of redissolvingand reprecipitating the HAA provides a purified, concentrated HAA forassay.

If the II, VII, IX and X concentrate is for laboratory or clinical use,the reserved superantant containing it is adjusted to a pH of about 5.2and the PEG concentration is raised, according to the molecular weightof the PEG used, e.g., 30 grams per 100 ml. with PEG-4000. Aftersuflicient mixing to dissolve the PEG and precipitate the concentrate,usually 30 minutes, the solution is centrifuged at about 10,000 RCF for10 minutes at room temperature. The precipitate is collected, washedwith 30 percent ethanol at 5 C. in buffer or in water and dissolved atneutral pH in a suitable buffer, e.g., sodium citrate-sodium chloride.Alternatively, Souliers method of precipitation and concentration withethanol may be used instead of PEG in the second precipitation step, butthe yield is moderately decreased with this procedure.

EXAMPLE 2 Concentration of HAA from gamma globulin, and/or preparationof gamma globulin free of hepatitis-associated antigen (HAA) forclinical use The starting material for this procedure can be FractronII+III (from Cohn Method VI), Fractions 11-3,

II-l, 2 and II (from Method X) or fractions obtained from similar orderivative techniques. The protein in these fractions is diluted in asuitable bufl er, e.g., glycinesodium citrate-sodium chloride at aboutneutral pH to a concentration of 12-22 mg./ ml. The temperature ismaintained at 15-25 C. The solution is adjusted to pH 3.0- 3.7 by slowlyadding acid, e.g., acetic, hydrochloric or citric acid, with mixing.Polyethylene glycol (PEG), 200-6000 molecular weight, is added to afinal concentration of 12-30 grams per 100 ml. of solution. The exactpercentage is increased for PEG of low molecular weight and decreasedfor materal of high molecular weight. After the PSG has been dissolvedby mixing and the HAA has been precipitated, which usually requires 30minutes, the solution is centrifuged at room temperature for at least 10minutes at a relative centrifugal force of about 10,000, and thesupernatant is separated cleanly from the HAA precipitate by decantationor vacuum aspiration and reserved.

The precipitate is reconstituted with three voldmes of buffer or salinesolution, and the walls of the centrifuge bottle are washed, bringingthe total volume to about 10 percent of the starting material, or to avolume which readily permits transfer of the reconstituted precipitatefrom the large centrifuge tube to a small one. The HAA is thenreprecipitated from the buffer or saline solution with 12-30 percent PEG2006000 and centrifuged. Since the precipitate contains the HAA, specialcare must be taken with this separation and with disposal of theprecipitate after it has been reconstituted in 0.2-1.0 ml. water,saline, or buffer, and assayed. This optional procedure of redissolvingand reprecipitating the HAA provides a purified, concentrated HAA forassay.

If the gamma globulin recovered is for laboratory or clinical use, thereserved supernatant containing it is adjusted to about pH 7.0 by slowlyadding a base, e.g., sodium hydroxide, with mixing. The precipitate iscollected, washed with 20-40 percent ethanol at -5 to l0 C. in buffer orin water and then dissolved in buifer, usually a glycine-sodiumcitrate-saline buffer at pH 7.0. As an alternative, this procedure canbe introduced into Method 6 after supernatant I has been collected andjust before the precipitation of Fraction II-III, or introduced intoMethod 9 after collection of Supernatant HI.

EXAMPLE 3 Concentration of HAA from albumin and/or preparation ofalbumin free of hepatitis-associated antigen (HAA) for clinical use Thestarting material for this procedure can be Cohn supernatant V orFraction V precipitate (from Method 6), or fractions obtained fromderivative techniques or other methods. The protein in these fractionsis diluted in a solvent, e.g., 0.9 percent sodium chloride, to a finalconcentration up to 50-60 mg./ml. and adjusted to about pH 7.0 by slowlyadding acid or base, e.g., hydrochloric, citric 0r acetic acid, orsodium hydroxide. The temperature of this solution is maintain at 15-20C. Polyethylene glycol (PEG), molecular weight 2006000, is added to afinal concentration of 12-30 grams per 100 ml. The exact percentage isincreased for the low-molecular-weight PEG and decreased for thehigh-molecular-weight material. After sufiicient mixing to dissolve thePEG and precipitate the HAA, usually 30 minutes, the solution iscentrifuged at room temperature for at least minutes at a relativecentrifugal force of about 10,000, and the supernatant is separatedcleanly from the HAA precipitate by decantation or vacuum aspiration andreserved.

The precipitate is reconstituted with three volumes of buffer or salinesolution, and the walls of the centrifuge bottle are washed, bringingthe total volume to about 10 percent of the starting material, or to avolume which readily permits transfer of the reconstituted precipitatefrom the large centrifuge tube to a small one. The HAA is thenreprecipitated from the buffer or saline solution with 12-30 percent PEG200-6000 and centrifuged'Since the precipitate contains the HAA, specialcare must be taken in this separation and in disposal of the precipitateafter it has been reconstituted in 0.2-1.0 ml. of water or salinesolution and assayed. This optional procedure of redissolving andreprecipitating the HAA provides a purified, concentrated HAA for assay.

If the albumin is for laboratory or clinical use, the pH of the reservedsupernatant containing it is adjusted to approximately 4.8 by slowlyadding an acid, e.g., acetic, hydrochloric or citric acid. Theprecipitate is collected washed with 40 percent ethanol in water or 0.9percent saline at -5 C., and a suitable solvent is added, e.g., water or0.9 percent sodium chloride. As an alternative, the Fraction Vprecipitate can be further fractionated by Method 6 and the resultingalbumin can then be processed as described above.

EXAMPLE 4 Concentration of HAA for assay from fibrinogen or AHFconcentrates and/ or preparation of fibrinogen or AHF concentrates freeof hepatitis-associated antigen (HAA) for clinical use Freshly prepared,fresh frozen or lyophilized fibrinogen or AHF concentrate prepared forclinical usereconstituted in sterile water or a suitable buifer-isdiluted to a protein concentration or 2-12 mg./ml. with physiologicsaline solution, 0-.02 M tris-0.02 M citrate buffer, or other buffer.Urea is added to a final concentration of 2.0-5.0 M. The procedure maybe carried out at a high pH or at a low PH.

High pH method: The pH of the solution is adjusted to 9.8-10.5 with anysuitable base. After the diluted fibrinogen or AHF has been mixed for 30minutes at pH 9.8-10.5 at room temperature, PEG 2006000 molecular weightis added to a final concentration of 12-30 grams/ ml. and mixing iscontinued for 30-120 minutes at room temperature to dissolve the PEG andprecipitate the HAA. The pH must be adjusted at this stage to preventany precipitation of the fibrinogen or AHF, or to dissolve any that hasalready precipitated. Such precipitation may occur at pH 10.0 or lower,but rarely above pH 10.3.

Low pH method: The pH of the solution is adjusted to 3.0-4.5 with anysuitable acid. After the diluted fibrinogen or AHF has been mixed for 30minutes at room temperature, PEG 200-6000 molecular weight is added to afinal concentration of 12-30 grams/ml. and mixing is continued for30-120 minutes at room temperature. The pH must be adjusted at thisstage to prevent any precipitation of the fibrinogen or AHF, or todissolve any that has already precipitated. Such precipitation may occurat a pH of 4.0 or more, but rarely below 4.2.

With either the high or low pH method, the mixture is then centrifugedfor at least 10 minutes at about 10,000 RCF at 20 C. to bring down theHAA, and the supernatant is aspirated carefully to avoid disturbing anyminute quantities of precipitate that may be visible at the bottom ofthe centrifuge cup or tube and reserved. The precipitate isreconstituted with three volumes of buffer or saline solution, and thewalls of the centrifuge bottle are washed, bringing the total volume toabout 10 percent of the starting material, or to a volume which readilypermits transfer of the reconstituted precipitate from the largecentrifuge tube to a small one. The HAA is then reprecipitated from thebuffer or saline solution with 12- 30 percent PEG 2006000, andcentrifuged at pH 9.8- 10.5 for the high pH method and pH 3.0-4.5 forthe low pH method, as described above. The final precipitate isdissolved in 0.2-l.0 ml. of saline, water or tris-citrate or otherbuffer for optimal concentration and then assayed. This optionalprocedure of redissolving and reprecipitating the HAA provides apurified, concentrated HAA fOr assay.

The fibrinogen or AHF in the aspirated reserved supernatant may beprecipitated by adding suflicient NaOH or HCl to bring the pH to 6.0.The precipitate is collected, washed with 10 percent ethanol in water at2 C. and a suitable solvent is added, e.g., 0.02 M tris citrate plus 0.1M NaCl.

EXAMPLE 5 Concentration of HAA from a plasma fraction containingprothrombin complex (Factors 11, VII, IX and X) and preparation of thefraction free of hepatitis-associated antigen (HAA) for clinical usePartially purified prothrombin complex (500 mg), obtained by DEAE columnchromatography, was dissolved in 0.03 M glycine-0.0005 M sodiumcitrate-0.01 percent sodium chloride buffer, pH 7.0, and diluted withadditional buffer to an ionic strength of 0.003 and a proteinconcentration of 5.0 mgJml. The temperature of this solution wasmaintained at 25 C. Polyethylene glycol (PEG) molecular weight 4000 wasadded to a final concentration of 20 grams per 100 ml. After 30 minutesof mixing to dissolve the PEG and precipitate the HAA, the solution wascentrifuged at room temperature for at least minutes at a relativecentrifugal force of approximately 10,000, and the supernatant wasseparated cleanly from the HAA precipitate by vacuum aspiration andreserved.

EXAMPLE 6 The precipitate from Example 5 was reconstituted with three(10 ml.) volumes of saline, and the walls of the centrifuge bottle werewashed with an additional 5 ml., bringing the total volume to 35 ml.allof which was pooled in a small test tube. The HAA was thenreprecipitated from the saline at pH 7.0 with percent PEG- 4000 andcentrifuged. Since the precipitate contains the HAA, special care mustbe taken in this separation and in disposition of the precipitate afterit has been reconstituted in 0.2-1.0 ml. normal saline and assayed.

EXAMPLE 7 The reserved supernatant from Example 5 containing Factors II,XII, XI and X was adjusted to a pH of 5.2 by adding 1 N hydrochloricacid, and the PEG concentration was raised to 30 grams per 100 ml. withPEG- 4000. After 30 minutes of mixing to dissolve the PEG andprecipitate the HAA, the solution was centrifuged at a RCF of 10,000 forat least 10 minutes at room temperature. The precipitate was collected,washed with 30 percent ethanol at 5 C. in buffer (pH 5.2) and dissolvedin 20 ml. of 0.3 M glycine-0.005 M sodium citrate-0.1 percent sodiumchloride buffer, at pH 7.0.

EXAMPLE 8 Concentration of HAA from gamma globulin and/or preparation ofgamma globulin free of hepatitis-associated antigen (HAA) for clinicaluse Fraction 11-3 (500 ml.) from Cohn Method 9, containing 166 mgprotein/mL, was diluted in 0.3 M glycine- 0.005 M sodium citrate-0.1percent sodium chloride buffer at pH 7.0 to a protein concentration of22 mg./ml. The temperature was maintained at 25 C. The solution wasadjusted by pH 3.5 by slowly adding 1 N hydrochloric acid, with mixing.Polyethylene glycol (PEG), 4000 molecular weight, was added to a finalconcentration of 20 grams per 100 ml. of solution. After minutes ofmixing to dissolve the PEG and precipitate the HAA, the solution wascentrifuged at room temperature for at least 10 minutes at a relativecentrifugal force of 10,000, and the supernatant was separated cleanlyfrom the HAA precipitate by vacuum aspiration and reserved.

12 EXAMPLE 9 The precipitate from Example 8 was reconstituted with three(10 ml.) volumes of saline solution, and the walls of the centrifugebottle were washed with 5 ml. of saline bringing the volume to 35 ml.allof which was transferred to a small test tube. The pH was adjusted to7.0 and the HAA was then reprecipitated from the saline solution with 20percent PEG-4000. Since the precipitate contains the HAA, special caremust be taken with this separation and the disposal of the precipitateafter it has been reconstituted in 1.0 ml. saline solution and assayed.

EXAMPLE 10 The reserved supernatant from Example 8 containing gammaglobulin was adjusted to pH 7.0 by slowly adding 1 N sodium hydroxide,with mixing. The precipitate was collected, washed with ethanol at 5 C.in buffer and dissolved in 500 ml. of 0.3 M glycine-0.005 M sodiumcitrate-0.1 percent butter, at pH 7.0.

EXAMPLE l1 Concentration of HAA from albumin and/ or preparation ofalbumin free of hepatitis-associated antigen (HAA) for clinical useFraction V precipitate (2.5 grams) from Cohn Method 6 was diluted in 0.9percent sodium chloride, to a final protein concentration of 50 mg./ml.and adjusted to pH 7.0 by slowly adding 1 N hydrochloric acid. Thetemperature of this solution was maintained at 25 C. Polyethylene glycol(PEG), molecular weight 4000, was added to a final concentration of 20grams per ml. After 30 minutes of mixing to dissolve the PEG andprecipitate the HAA, the solution was centrifuged at room temperaturefor 10 minutes at a relative centrifugal force of 10,000, and thesupernatant was separated cleanly from the HAA precipitate by vacuumaspiration and reserved.

EXAMPLE 12 The precipitate from Example 11 was reconstituted with three(10 m1.) volumes of saline, and the walls of the centrifuge bottle werewashed with 5 ml. bringing the total volume to 35 ml.all of which wasthen pooled in a small test tube. The pH was adjusted to 7.0 and the HAAwas then reprecipitated from the saline with 20 percent PEG-4000 andcentrifuged. Since the precipitate contains the HAA, special care mustbe taken in this separation and in the disposal of the precipitate afterit has been reconstituted in 1.0 ml. of saline solution and assayed.

EXAMPLE 13 The pH of the reserved supernatant from Example 11 containingthe albumin was adjusted to 4.8 by slowly adding 1 N hydrochloric acid,and the PEG concentration was raised to 30 grams per 100 ml. with PEG4000. After 30 minutes of mixing to dissolve the PEG and precipitate theHTA, the solution was centrifuged at a RCF of 10,000 for at least 10minutes at room temperature. The precipitate was collected, washed with40 percent ethanol in 0.9 percent saline at 5 C., and dissolved in 500ml. of 0.9 percent sodium chloride, at pH 7.0.

EXAMPLE l4 Concentration of HAA for assay from fibrinogen or AHFconcentrates and/or preparation of fibrinogen or AHF concentrates freeof hepatitis-associated antigen (HAA) for clinical use Lyophilizedfibrinogen or fibrinogen-rich AHF concentrate prepared for clincial use(approximately l' /2-2 gm.) was reconstituted in 200 ml. of distilledwater and diluted to a protein concentration of 2 mg./ml. with 0.02 Mtris- 0.02 M citrate buifer, pH 7.0. Urea was added to a finalconcentration of 2.5 M.

High pH method: The pH of the solution was adjusted to 10.3 with 1 NNaOH. After the diluted fibrinogen or 13 AHF had been mixed for 30minutes at room temperature, PEG-4000 was added to a final concentrationof 30 grams per 100 ml. and mixing was continued for 30 mintues. The pHwas maintained at 10.3 throughout this stage to prevent anyprecipitation of the fibrinogen or AHF, and to dissolve any that hadalready precipitated.

EXAMPLE 15 Low pH method: The procedure of Example 14 was followed, butthe pH was adjusted to 3.5 using 1 N HCl. After the diluted fibrinogenor AHF had been mixed for 30 minutes at room temperature, PEG-4000 wasadded to a final concentration of 20 grams per 100 ml. and mixing wascontinued for 30 minutes at room temperature. The pH was maintained at3.5 throughout this stage to prevent any precipitation of the fibrinogenor AHF, and to dissolve any that had already precipitated. Suchprecipitation may occur at a pH of 4.0 or more, but rarely below 4.2.

EXAMPLE 16 With both the high and low pH methods, of Examples 14 and 15,the mixture was then centrifuged for about one hour at 10,000 RCF at 20C. to bring down the HAA, and the supernatant was aspirated carefully toavoid disturbing any minute quantity of precipitate visible at thebottom of the centrifuge cup. The precipitate was reconstituted withthree successive 10 ml. volumes of saline, and the walls of thecentrifuge bottle were washed with an additional ml. bringing the totalvolume to 35 ml.all of which was pooled in a small test tube. The HAAwas then reprecipitated from the saline solution with 30 percentPEG-4000, at pH 7.0 and centrifuged. The final precipitate was dissolvedin 1.0 ml. of 0.02 M tris-0.02 M citrate buffer for optimalconcentration.

EXAMPLE 17 The fibrinogen or AI-IF in the aspirated supernatant fromExample 16 was precipitated by adding sufiicient 1 N NaOH for the low pHmethod or 1 N HCl for the high pH method to bring the pH to 6.0. It wasthen centrifuged at an RCF of 10,000 for at least minutes at roomtemperature, the precipitate collected and washed with 10 percentethanol in water at 2 C. and dissolved in 200 ml. of 0.4 percentcitrate-0.9 percent NaCl, at pH 7.0.

EXAMPLE 18 Concentration of HAA from a plasma fraction containingprothrombin complex (Factors II, VH, IX and X) and extraction of thefraction free of hepatitis-associated antigen (HAA) for clinical usePrecipitated or lyophilized partially purified prothrombin complex (500mg), obtained by DEAE column chromatography, was dissolved in a 20percent PEG- butfer solution of 0.03 M glycine-0.0005 M sodiumcitrate-0.01 percent sodium chloride, pH 7.0 (ionic strength of 0.003),to a protein concentration of 5.0 mg./ ml. The temperature of thissolution was maintained at 25 C. After 30 minutes of mixing to extractthe prothrombin complex and precipitate the HAA, the solution wascentrifuged at room temperature for at least 10 minutes at a RCF ofapproximately 10,000, and the supernatant was separated cleanly from theHAA precipitate by vacuum aspiration and reserved.

EXAMPLE 19 The precipitate from Example 18 was reconstituted with three(10 ml.) volumes of saline, and the walls of the centrifuge bottle werewashed with an additional 5 ml., bringing the total volume to 35 ml.allof which was pooled in a small test tube. The HAA was thenreprecipitated from the saline at pH 7.0 with 20 percent PEG- 4000 andcentrifuged. Since the precipitate contains the HAA, special care mustbe taken in this separation and in disposition of the precipitate afterit has been reconstituted in 0.1-1.0 ml. normal saline and assayed.

14 EXAMPLE 20 The reserved supernatant from Example 18 containingFactors II, VII, IX and X was adjusted to a pH of 5.2 by adding 1 Nhydrochloric acid, and the PEG concentration was raised to 30 grams perml. with PEG-4000. After 30 minutes of mixing to dissolve the PEG andprecipitate the HAA, the solution was centrifuged at a RCF of 10,000 forat least 10 minutes at room temperature. The precipitate was collected,washed with 30 percent ethanol at 5 C. in buffer (pH 5.2) and dissolvedin 20 ml.- of 0.03 M glycine-0.005 M sodium citrate-0.1 percent sodiumchloride buffer, at pH 7.0.

It should be understood that reference to the isoelectric point of asubstance means the pH at which the net charge on a molecule in solutionis 0. At this pH, amino acids exist almost entirely in the zwitterionstate; that is, the positive and negative groups are equally ionized. Asolution of proteins or amino acids at the isoelectric point exhibitsminimum conductivity, osmotic pressure, and viscosity.

It is also understood that saline solution, unless otherwise indicated,refers to physiologic saline solution.

It should be apparent from the foregoing detailed description that theobjects set forth hereinabove have been successfully achieved. Moreover,while there is shown and described a present preferred embodiment of theinvention, it is to be distinctly understood that the invention is notlimited thereto but may be otherwise variously embodied and practicedwithin the scope of the following claims.

What is claimed is:

1. A method of separating heptatitis-associated antigen from a solutioncomprising a solvent which is physiologically tolerable on injection,the hepatitis-associated antigen, and other proteins contained in aplasma fraction selected from the group consisting of albumin, gamma,globulin, coagulation Factors II, V, VII, IX, X, XI and XIII,fibrinogen, antihemophilic factor, plasminogen, cerloplasmin,transferrin, thyroxin-binding protein, antithrombin HI, a antitryspin, amacroglobulin, Ci inactivator, inter a-trypsin inhibitor and mixturesthereof, so as (1) to enable the remaining plasma fraction to betransfused with reduced likelihood of transmitting heptatitis to therecipient, or (2) to concentrate the antigen for assay withsubstantially increased antigen/other proteins ratio relative to theantigen/other proteins ratio in the original solution, or (3) toaccomplish both (1) and (2) simultaneously, comprising the steps of:

(a) maintaining the pH of said solution within a range removed as far aspossible from the isoelectric point of said other proteins withoutcausing denaturation and still permitting precipitation of said antigen;

(b) adding polyethylene glycol having a molecular weight of from about200 to about 6,000 to a concentration of from about 12 to about 30 gramsper 100 milliliters of said solution to thereby obtain a precipitateconsisting essentially of said antigen, said other proteins remaining inthe supernatant substantially free of said antigen; and

(c) separating said precipitate from said supernatant.

2. A method of separating hepatitis-associated antigen from a solutioncomprising a solvent which is physiologically tolerable on injection,the hepatitis-associated antigen and other proteins contained in aplasma fraction selected from the group consisting of albumin, gammaglobulin, coagulation Factors II, V, VII, IX, X, XI and XIII,fibrinogen, antihemophilic factor, plasminogen, ceruloplasmin,transferrin, thyroxin-binding protein, antithrombin IH, a antitryspin, amarcroglobulin, Ci inactivator, inter a-trypsin inhibitor and mixturesthereof, so as (l) to enable the remaining plasma fraction to betransfused with reduced likelihood of transmitting hepatitis to therecipient, or (2) to concentrate the antigen for assay withsubstantially increased antigen/other proteins ratio relative to theantigen/other proteins ratio in the original solution, or (3) toaccomplish both (1) and (2) simultaneously, comprising the steps of:

(a) maintaining the pH of said solution within a range sulficientlyclose to the isoelectric point of at least some of said other proteinsand said antigen to permit precipitation thereof;

(b) adding polyethylene glycol having a molecular weight of from about200 to about 6,000 to a concentration of from about 12 to about 30 gramsper 100 milliliters of said solution to thereby obtain a precipitatecontaining said antigen and at least some of said other proteins;

() separating said precipitate from its supernatant;

(d) extracting said other proteins from said antigen in said precipitatewith a physiologically tolerable solvent having a pH removed from theisoelectric point of said other proteins to produce a supernatantsubstantially free of said antigen, said antigen remaining in theprecipitate; and

(e) separating said precipitate consisting essentially of said antigenfrom said supernatant containing said other proteins.

3. A method as defined in claim 2, wherein said polyethylene glycol hasa molecular weight of about 4,000 and said concentration is from about20 to 30 grams per 100 milliliters of solution.

4. A method as defined in claim 2, wherein said steps are performed at atemperature of from about 15 to 25 C.

5. A method as defined in claim 4, wherein said steps are performed atroom temperature.

6. A method as defined in claim 2, wherein said solvent has an ionicstrength between 0.001 and 0.2.

7. A method as defined in claim 6, wherein said solvent is selected fromthe group consisting of glycine-citratesaline butler, tris(hydroxymethylaminomethane-citrate =buifer, tris(hydroxymethylaminomethane-citrate-urea buffer, phosphate bufier, phosphate-salinebutler, ammonium or sodium acetate, sodium bicarbonate-CO an amino acid,physiologic saline solution, and water 8. A method as defined inclaim'7, wherein said solvent is a buffer solution of a concentrationsuitable to provide a predetermined pH.

9. A method as defined in claim 2, wherein said antigen is removed fromsaid protein by centrifugation or filtration.

10. A method as defined in claim 9, wherein said centrifugation variesfrom about 10,000 to 15,000 RCF for about 4 minutes to about 1 hour.

11. A method as defined in claim 9, wherein said separation is byfiltration using a filter having a pore size below about 0.6 mg.

12. A method as defined in claim 11, wherein said pore size is fromabout 0.45 to about 0.2 m

13. A method as defined in claim 2, further comprising purifying andconcentrating said antigen by the steps of:

(a) maintaining solubility of said antigen in a solvent at a pH of about7;

(b) adding polyethylene glycol having a molecular weight of about 4,000to a concentration of about 20 percent to reprecipitate said antigen;and

(c) removing said reprecipitated antigen from the resulting supernatant.

14. A method as defined in claim 2, further comprising concentratingsaid protein by:

(a) adjusting the pH of said supernatant to near the isoelectric pointof said protein to thereby precipitate said protein; and

(b) collecting said precipitated protein.

15. A method as defined in claim 2 wherein said plasma fractioncomprises prothrombin complex.

16. A method as defined in claim 1, wherein said polyethylene glycol hasa molecular weight of about 4,000 and said concentration is from about20 to 30 grams per milliliters of solution.

17. A method as defined in claim 1, wherein said steps are performed ata temperature of from about 15 to 25 C.

18. A method as defined in claim 17, wherein said steps are performed atroom temperature.

19. A method as defined in claim 1, wherein said solvent has an ionicstrength between 0.001 and 0.2.

20. A method as defined in claim 19, wherein said solvent is selectedfrom the group consisting of glycinecitrate-saline buffer,tris(hydroxymethyl) aminomethanecitrate buffer, tris(hydroxymethyl)aminomethane-citrateurea buffer, phosphate buffer, phosphate-salinebufier, ammonium or sodium acetate, sodium bicarbonate-CO an amino acid,physiologic saline solution, and water.

21. A method as defined in claim 19, wherein said solvent is a buttersolution of a concentration suitable to provide a predetermined pH.

22. A method as defined in claim 1, wherein said antigen is removed fromsaid protein by centrifugation or filtration.

23. A method as defined in claim 22, wherein said centrifugation variesfrom about 10,000 to 15,000 RCF for about 4 minutes to about 1 hour.

24. A method as defined in claim 22, wherein said separation is byfiltration using a filter having a pore size below about 0.6 mg.

25. A method as defined in claim 24, wherein said pore size is fromabout 0.45 to about 0.2 mg.

26. A method as defined in claim 1, further comprising purifying andconcentrating said antigen by the steps of:

(a) maintaining solubility of said antigen in a solvent at a pH of about7;

(b) adding polyethylene glycol having a molecular weight of about 4,000to a concentration of about 20 percent to reprecipitate said antigen;and

(c) removing said reprecipitated antigen from the resulting supernatant.

27. A method as defined in claim 1, further comprising concentratingsaid protein by:

(a) adjusting the pH of said supernatant to near the isoelectric pointof said protein to thereby precipitate said protein; and

(b) collecting said precipitated protein.

28. A method as defined in claim 1, wherein said plasma fractioncomprises prothrombin complex.

References Cited UNITED STATES PATENTS 3,415,804 12/1968 Poison 260-1123,652,530 4/1972 Johnson et al. 260-112 3,630,840 12/1971 Wagner 260-412OTHER REFERENCES General Biochemistry, 1958, Fruton et al. pp. 101-102.

HOWARD E. SCHAIN, Primary Examiner US. Cl. X.R.

